Sequence-specific detection of very low quantities of DNA or RNA is useful for a wide range of applications, including clinical diagnostics, food safety testing, forensics, or environmental microbiology.
More generally, most biological species of interest, notably proteins, polysaccharides, nucleic acids, phospholipids, and the combination of such, are charged in solution, and thus constitute ions or most often macroions, meaning they bear a multiplicity of charges. This is also true for numerous colloids or cells or organelles, including, in a non-exhaustive way, viruses, cell nuclei, endosomes, exosomes, mitochondria, bacteria, vesicles. Macroions are also often encountered in chemistry, e.g. as latexes, colloids, nano or microparticles, nanorods, fibers, charged polymer, polyelectrolytes, vesicles, micelles. The charge of these species may be a convenient way to detect said species, since it is an intrinsic property of said species, and does not impose an additional step of labeling.
Charge may be used as a means to separate species, like in the known methods of electrophoresis, electrochromatography or isotachophoresis. However, the known charge-based methods of species detection may not be very sensitive and may also lack specificity, since biological or chemical buffers also contain in general numerous small ions which create a high conductivity background.
Enzymatic amplification methods have provided a tremendous potential in sensitivity, and Polymerase Chain Reaction (PCR) in particular, has become a major and routine tool for genetic analysis.
Numerous systems now exist, from benchtop machines costing a few thousands of , to more elaborate and high throughput quantitative PCR machines costing several tens of k. Most of these systems, however, use fluorescence-based detection, and remain dependent on electric power supply from the mains.
Important applications, regarding e.g. pathogen detection in remote environments, biosafety or forensics, would demand portable “point of care” or “point of sampling” assays, and thus efforts over the last decade have been directed in order to integrate this type of assay into microfluidic systems, as described e.g. in A. K. White et al., Proceedings of the National Academy of Sciences, 2011, 108, 2-7.
In order to achieve this, different strategies were proposed which aimed either at reducing the power consumption of fluorescence-based PCR (P. J. Asiello and A. J. Baeumner, Lab on a chip, 2011, 11, 1420-30.) e.g. using diode technologies, or using a DNA equivalent of immuno-agglutination (J. Li, H. Alshammari et al, proc. Microtas 2011, CBMS Publ., pp. 1959-1961, or more radically at avoiding any optics by electrochemistry (see e.g. B. S. Ferguson et al, Analytical chemistry, 2009, 81, 7341-7346.
Fluorescence detectors are so far still unchallenged in terms of sensitivity. However fluorescent detection requires labelling reagents, and developing very low cost technologies, notably for the developing world, is still challenging (see e.g. P. Yager et al., Nature, 2006, 442, 412-8.).
It would thus be very interesting to provide methods able to detect and monitor nucleic acid amplification, and more generally, macroions of biological, medical, environmental, forensic, or chemical interest, without using labels or costly detection techniques. Unfortunately, this is not possible in the state of the art, because the amplification of nucleic acids does not change the global conductivity of a solution. Some electrochemical methods exist, as recited e.g. in Deféver T et al, J Am Chem Soc. 2009 Aug. 19; 131(32):11433-41, but they require labels. It is thus a first object of the invention, to provide a method to detect microions, and particularly to monitor the amplification of nucleic acids, without label and with direct electric read, e.g. conductimetric.
In addition, the conventional methods of quantitative PCR can only be applied to relatively short fragments (less than 2 kbp (see e.g: M. Stegger et al., Clinical Microbiology and Infection, 18: 395-400. doi: 10.1111/j.1469-0691.2011.03715.x). A number of new amplification methods increasingly used in research, including long range PCR (e.g. O. Harismendy et al., Genome biology, 2009, 10, R32), or isothermal amplification produce long nucleic acids fragments. A non-exhaustive list of nucleic acid amplification methods are reviewed and listed e.g. in A. Niemz et al; Trends in Biotechnology, May 2011, Vol. 29, No. 5, pp 240-250. However, these new methods are seldom used in diagnosis or routine, because either of their complexity, or their lack of quantitativeness.
Large nucleic acid molecules can also be analyzed by electrophoresis. The well-known method used to separate large nucleic acids is pulse-field electrophoresis in gels, as described e.g. in WO8402001 to Schwartz and Cantor. This method, however, is very time consuming, e.g., typically 24 hours for a separation, labor intensive, and requires a lot of material.
Attempts have been made to transpose this to capillary electrophoresis, but as shown in Mitnik et al. Science, 1995, 267, 5195, 219-22, high electric fields applied to macroions solutions in capillary lead to another electrokinetic phenomenon, different from the normal transport of ions along field lines. This phenomenon is a nonlinear electrohydrodynamic instability, which gathers DNA into aggregates, creates a lot of noise and ruins separation. This phenomenon is highly non-linear, and its inception depends on field frequency, field strength, and on the concentration and size of the nucleic acid.
Attempts using amphoteric buffers to suppress these aggregates, which is for capillary electrophoresis a strong nuisance, have been proposed e.g. in Magnusdottir et al. Biopolymers, 49, 385-401, (1999) but even then the electric field has to be decreased as compared to conventional capillary electrophoresis, and separation times are too long.
Besides this limitation, capillary electrophoresis systems generally use optical detection methods, either based on UV absorption, or on Laser Induced Fluorescence (LIF), which are expensive, bulky and have a large power consumption. Therefore, attempts have been made to replace these detection methods by direct conductivity detection, since the species separated in electrophoresis are in general, charged.
Numerous methods for conductivity detection, notably in the context of electrokinetic separation and analysis methods, such as capillary electrophoresis, microchannel electrophoresis, or isotachophoresis, have been proposed in the literature.
Reviews can be found for instance in V. Solinova et al., J. Sep. Sci. 2006, 29, 1743-1762 and R. M. Guijt et al., Electrophoresis 2004, 25, 4032-4057. Conductivity detection requires at least two electrodes, in electric connection with the medium under study. Typically, conductivity detection can be implemented in two different families, contact detection, in which the electrodes are in direct electric connection, meaning that they can conduct through the solution continuous or alternating current, or contactless detection, in which said electrodes are in electric relation with the solution through a dielectric layer, so that it can conduct only or mainly alternating current. Contactless conductivity measurements rely on high excitation frequencies (typically in the kHz or MHz range) and capacitive coupling between the electrodes and the solution. The frequency at which conduction occurs typically depends on the thickness of the dielectric layer. This method has the advantage of placing the electrodes outside of the solution through a dielectric, minimizing interferences from the (DC) high electric field, and ground loops. For moderately to highly conductive solutions, however, it is limited in sensitivity, because the impedance of the dielectric layer is high as compared to that of the solution. In addition, the high excitation frequencies required to keep the dielectric's impedance at a reasonable value lead to more expensive and bulky instrumentation.
Contact conductivity measurements uses electrode-solution contact to make measurements of the solution conductivity. This approach is more sensitive than contactless detection, but in methods involving a strong electric fields for moving the species of interest, and notably in capillary electrophoresis, microchannel electrophoresis, it is prone to interactions between the separation field and the detection electronics, resulting in unwanted electrochemical reactions, electrolysis of water, bubble formation and increased noise. To avoid this, Prest et al., in Analyst, 2002, 127, 1413-1419, propose a contact based detection, but they need to have the measurement electrodes in separate vials distant from the separation channel, which reduces the sensitivity. Mo et al., Anal. Commun, 1998, 35, 365-367, also discloses a system, in which electric insulation is performed by an optocoupler, but all these electronic systems have some leaks, and the sensitivity remains low, in the mM range.
Documents MILES US 2002/0070114 and US 2005/0136466 and BRYNING US 2010/0203580 are also known which teach detection methods wherein an analyte is trapped in an electric field.
There are thus needs to improve the sensitivity of conductimetric detection in the presence of an external stimulating field. A need also exists to obtain a low-cost, portable detection technology, notably for analytes at low concentrations, allowing evolution from a “chip in the lab” to a “lab on a chip” paradigm.
A need also exists for a label-free direct-reading of the presence of macroions in a solution, preferably nucleic acids and notably DNA as such as obtained with new amplification methods.
A need also exists to provide a label-free method to detect macroions, and in particular to monitor the amplification of nucleic acids.
A need also exists to obtain a new, simple and low cost electronic device, able to ensure satisfying contact conductivity measurement in microchips with high sensitivity even when a relatively conductive buffer and high external stimulating field are used.
The present invention aims to meet one or more of the aforementioned needs.